Current vaccination strategies are predicated on eliciting expansion of a pre-existing pool of mature peripheral T lymphocytes. We intend to develop an entirely different methodology, termed thymic vaccination, which will use peptide-based analogs of cognate antigens to shape the T cell repertoire during T lineage development. Recent work in T cell receptor (TCR) transgenic (tg) systems has shown that focal local structural alterations of peptide-major histocompatibility (pMHC) complexes affected by conservative modification at the central peptide residue(s) of a cognate peptide and accompanying compensatory structural modifications around this site can create a powerful stimulus for positive selection. This selection results in differentiation and exportation of mature thymocytes. To precisely define the rules governing the relationship between cognate antigens and positively selecting stimuli, we will examine the C57BL/6 (H-2b) response to a variety of pathogens using structural and functional approaches. First, high energy synchrotron radiation and ultrafast automated protein structure determinations will be employed to determine structures of 200-300 Kb and Db pMHCI complexes per year. The latter will include altered peptide ligand (APL) variants mutated at the central TCR contact residues as well as secondary sites, including chemically synthesized organic molecules and biophysical NMR-derived data. This structural information will be correlated with functional data in normal as well as TCR transgenic B6 mice raised under gnotobiotic conditions to define the precise nature of variants that lead to positive selection. Second, we shall develop individual pathogen libraries and search engines for identifying MHC class I and II epitopes within the genome of an infectious agent. Third, we will test whether thymic vaccination can be utilized to elicit protective immunity in a number of murine infectious models including vesicular stomatitis virus (VSV), lymphocytic choriomeningitis virus (LCMV) and influenza A. T cell protection resulting from generation of new TCR specificities against viral determinants not previously recognized by B6 mice should be demonstrable.